The invention concerns in general a spectroscopic measuring method using optical converters to detect changes of optical thickness in optically-active parts of such a converter as e.g. occur in reflectrometric interference spectroscopy in the fields of chemical or biochemical analysis, and especially in the field of microfluid laboratory measuring systems. The invention concerns in particular a method for the spectroscopic measurement of an optophysical quantity or its change over time using the cited optical converter to convert the optophysical quantity into a modulated (especially frequency or phase-modulated) signal. The converter is conventionally transilluminated with electromagnetic radiation (especially in the visible range) for a period of t greater than =0, whereby the electromagnetic radiation varies within a wavelength or frequency range, and a corresponding modulated spectrum is determined from this, and whereby the performance quantities of the interference spectroscopy measuring setup, especially of the converter and a radiation source that generates the electromagnetic radiation, are subject to temporal fluctuations.
For a number of decades, chemical and biochemical sensors of the cited type have been used for research and commercial use. Optical measuring procedures are of particular interest that allow contact-free and hence non-destructive sensing of the relevant optical quantities. These methods are particularly distinguished in that they can be carried out in situ on the samples to be investigated, especially remote controlled. In particular, these methods are increasingly being used for microfluid laboratory measuring systems in which laboratory microchips are used. Such a microchip is e.g. described in detail in the patent application by the present applicant with the title, xe2x80x9cDevice to Operate a Laboratory Microchipxe2x80x9d (official application number DE 199 28 410.5), in particular in FIGS. 1 and 2 to which full reference is made in the present context.
Reflectrometric interference spectroscopy (RifS) is a standard method for characterising structural properties of a surface, and it has been recognised as a fundamental optical converter principle in the field of chemistry or biosensor technology since the early nineties. This procedure uses the interference of white light reflected on a thin, non-absorbing optical layer. The spectral distribution of the reflected light intensity is measured, whereby the respective film thickness is determined by evaluating the obtained interference spectrum. The evaluation is carried out with a high degree of precision by determining the respective wavelength of the interference peaks and their shift in relation to the wavelength in relation to the film thickness.
With these prior-art sensors, the film thickness is determined from a spectrum measured in a reflection or transmission by evaluating the position of one or more base points of the spectrum, e.g. curve extremities (maximum, minimum, or points of inflection), and calculated via a curve computationally adapted to the respective spectrum. Such a measuring setup is e.g. disclosed in DE 4 200 088 C2. Alternately to the evaluation in the spatial domain described therein, the spectra can be evaluated using changes in a modulation frequency of a corresponding signal in the pulse domain, i.e., in the frequency domain whereby prior-art methods of Fourier transformation are used. A corresponding method is e.g. described in an article by G. Kraus and G. Gauglitz: xe2x80x9cApplication and Comparison of Algorithms for Evaluation of Interferograms,xe2x80x9d in Fresenius J. Anal. Chem. 344, 153 (1992).
To obtain the interference spectrum required for evaluation from the measured intensity data, calibration is necessary is all the cited cases. This calibration is usually done by forming a quotient with a reference spectrum that is either done before the actual measurement using as a basis the measuring setup provided for the measurement, or during the actual experiment using an identical, similar or different measurement setup. The referencing is done either initially, i.e., once, or continuously during measurement by determining reference spectra.
The disadvantage of a single calibration done before the actual experiment is that any arising fluctuations during the experiment, e.g. in the spectral sensitivity of the measuring setup due to changes in the colour temperature of the utilised radiation source or the spectral sensitivity of the utilised optical components or their measuring sensitivity cannot be covered or compensated during measurement, and these fluctuations necessarily lead to the distortion of the measurement results.
In contrast, calibration or referencing done at the same time as the experiment has the disadvantage that a second, if possible identical measuring arrangement is required whereby the measuring beam is directed using a beam divider both in the actual measuring setup and in the reference measuring setup. In particular, the beam division worsens the signal-to-noise ratio since the beam intensity is reduced by half, and the overall quality of the measurement results is worsened.
Another problem inherent in the prior-art interference measuring methods is that the reference points (especially interference extrema) of the evaluated spectra produce a shift in the extrema when the performance quantities of individual components or the entire measuring setup change, and hence produce an apparent change in the measured path lengths or layer thicknesses.
The present invention is hence based on the problem of presenting a method of the initially-described type and a corresponding measuring setup that avoids the cited disadvantages of the state-of-the-art. To be avoided in particular are distortions of the measuring results due to changes in performance quantities of the measuring setup and a simultaneous worsening of the signal-to-noise ratio.
The problem is solved by the features of the independent claims. Advantageous embodiments are cited in the dependent claims.
The particular feature of the invention is that the intensity distribution measured at time t=0 is used as a reference spectrum for all other measurements (self-referencing), and the relative change (and not the measured quantity itself as is conventional) is determined for time t greater than 0 to identify the changes in the optical path length or optical thickness by evaluating the modulation amplitude of this xe2x80x9cdifferentialxe2x80x9d spectrum.
The concept on which the invention is based is in particular to differentiate whether the temporal changes of the modulated spectrum are changes in the path length or optical thickness to be determined in the respective measurement, or only fluctuations in the performance quantities of the measuring setup. A disturbance calculation is continuously made based on the assumed changes in the performance quantities to separate the actual measurement effects from the cited distortion of the measurement signals using the disturbance calculation.
Let it be noted that the term xe2x80x9clight opticsxe2x80x9d in the present context refers to the entire wavelength range between infrared (IR) and ultraviolet (UV).
In a preferred exemplary embodiment, a disturbance of the measured quantity is assumed to be a +bxcex that results in a shift xcex94xcex of an extremum to be expected at a point, according to the relationship:                     Δλ        =                  b                                    [                                                                    ∂                    2                                    ⁢                                                            R                      D                                        ⁡                                          (                      λ                      )                                                                                        ∂                                      λ                    2                                                              ]                                      λ              =                              λ                0                                                                        (        1        )            
Where RD(xcex) is the reflectivity depending on xcex, and where the position of the extremum is given by the following depending on the optical layer thickness D:                                           λ            0                    ⁡                      (            D            )                          =                              4            ⁢            D                    k                                    (        2        )            
(where k=order of the respective interference extremum). Depending on the available computing power, disturbance calculations of a higher order can be carried out.
In a development of the inventive idea, the following individual steps can be carried out during the procedure:
a) determination of the reference values of the modulated spectrum at t=0;
b) determination of the measured values, and a calculation of a xe2x80x9cdifferentialxe2x80x9d spectrum by forming a quotient with the reference values from a) for t greater than 0;
c) determination of a modulation amplitude of the spectrum calculated in step b);
d) calculation of a change in the optical-physical quantity from the modulation amplitude determined in step c).
In regard to determining the amplitude of the calculated modulated spectrum according to step c), mathematical relationships between the quantity to be measured and the beam intensity measured in transmission or reflection can be advantageously used. As an example, let us take an expression shown below in equation (3) that reflects the known mathematical relationship of the reflectivity of a thin optical layer under normal light at the point of incidence and slight reflection. From the modulation amplitude of the differential spectrum calculated in this manner, we can determine changes in the optophysical quantity to be measured as will later be discussed in detail.                                           R            D                    ⁡                      (            λ            )                          =                              R            1                    +                      R            2                    +                      2            ⁢                                                            R                  1                                ⁢                                  R                  2                                                      ⁢            cos            ⁢                          xe2x80x83                        ⁢                          (                                                4                  ⁢                  π                  ⁢                                      xe2x80x83                                    ⁢                  D                                λ                            )                                                          (        3        )            
In equation (3), D is the optical thickness of the layer, and quantities R1, R2 are the reflectivity of a substrate layer at the substrate/layer and layer/sample transitions. The amplitude of the differential spectrum is advantageously evaluated by adapting curves to a model function (e.g. a polynomial), whereby purely statistical noise suppression already exists. In addition, variable instrument quantities can be thereby suppressed.
The method suggested according to the invention can be advantageously used in a layer system in a microfluid microchip consisting of at least two, at least partially optically transparent layer components, whereby the optophysical quantity represents an optical path length according to the mathematical product consisting of the refraction index or indices of the respective layer component(s), and the respective physical length, especially the physical thickness of at least one of the layer components.
The invention will be described below with reference to exemplary embodiments using drawings. Other problems, advantages and features of the invention are found in the features of the patent claims.